Mammalian Muscle Cells Research Articles

Sarcoplasmic Reticulum Calcium Content in Normal and Dystrophic Mammalian Skeletal Muscle Fibers

The evaluation of the maximal amount of releasable Ca2+ (Ca2+-content) that is stored in the sarcoplasmic reticulum (SR) of skeletal muscle fibers is a topic of great importance in muscle physiology. We have developed a method to quantitatively estimate the SR Ca2+ content in isolated mammalian muscle fibers based on Ca2+ dependent fluorescence measurements performed in the presence of high concentrations of Ca2+ buffers, and model calculations of Ca2+ flux underlying the fluorescence changes. Single fibers were enzymatically isolated from murine FDB muscles and impaled with two microelectrodes. One electrode, filled with 1M KCl, was used to record the membrane potential throughout the procedure. The other electrode was used to dialyze the fiber with a solution containing either 6-15 mM BAPTA, or up to 60 mM EGTA (pCa=7.0), and adjusted to pH=7.0 with 20 or 60 mM MOPS, respectively. Free myoplasmic Ca2+ concentration ([Ca2+]) changes were measured with the low affinity indicator OGB-5N (200 μM). SR Ca2+ release was elicited by either 20 mM caffeine, or 1 mM 4-chloro-methy-phenol (4CmC), added to the external solution. The maximal fluorescence change of the Ca2+ indicator was assessed at the end of the protocol by exposing the fibers to saponin (0.1 mg/ml in isotonic CaCl2, pH=7.0 with 20 mM MOPS). To prevent changes in shape of the fibers under these conditions, they were pretreated for 1 min with 1% formaldehyde (in Tyrode). The experiments were conducted at 20 °C. A single model compartment was used to estimate (from the [Ca2+] changes) the total Ca2+ released in response to caffeine/4CmC application. We obtained values of SR Ca2+ content in the range of 15-27 mM for normal FDB fibers. Interestingly, comparable values were obtained in fibers from mdx mice.

Long-term and room temperature operable bioactuator powered by insect dorsal vessel tissue

We present a bioactuator powered by insect dorsal vessel tissue which can work for a long time at room temperature without maintenance. Previously reported bioactuators which exploit contracting ability of mammalian heart muscle cell have required precise environmental control to keep the cell alive and contracting. To overcome this problem, we propose a bioactuator using dorsal vessel tissue. The insect tissue which can grow at room temperature is generally robust over a range of culture conditions compared to mammalian tissues and cells. First, we confirm that a dorsal vessel tissue of lepidoptera larva Ctenoplusia agnata contracts spontaneously for at least 30 days without medium replacement at 25 degrees C. Using the dorsal vessel tissue cultured under the same conditions, we succeed in driving micropillars 100 microm in diameter and 1000 microm in height for more than 90 days. The strongest displacement of the micropillar top occurs on the 42(nd) day and is 23 microm. Based on these results, the contracting force is roughly estimated as 4.7 microN which is larger than that by a few mammalian cardiomyocytes (3.4 microN). Definite displacements of more than 10 microm are observed for 58 days from the 15(th) to the 72(nd) days. The number of life cycles can be roughly calculated as 7.5 x 10(5) times for the average frequency of about 0.15 Hz, which is no less than that of conventional mechanical actuators. These results suggest that the insect dorsal vessel tissue is a more promising material for bioactuators used at room temperature than other biological cell-based materials.

Muscle fatigue examined at different temperatures in experiments on intact mammalian (rat) muscle fibers

In experiments on small bundles of intact fibers from a rat fast muscle, in vitro, we examined the decline in force in repeated tetanic contractions; the aim was to characterize the effect of shortening and of temperature on the initial phase of muscle fatigue. Short tetanic contractions were elicited at a control repetition rate of 1/60 s, and fatigue was induced by raising the rate to 1/5 s for 2–3 min, both in isometric mode (no shortening) and in shortening mode, in which each tetanic contraction included a ramp shortening at a standard velocity. In experiments at 20°C (n = 12), the force decline during a fatigue run was 25% in the isometric mode but was significantly higher (35%) in the shortening mode. In experiments at different temperatures (10–30°C, n = 11), the tetanic frequency and duration were adjusted as appropriate, and for shortening mode, the velocity was adjusted for maximum power output. In isometric mode, fatigue of force was significantly less at 30°C (∼20%) than at 10°C (∼30%); the power output (force × velocity) was >10× higher at 30°C than at 10°C, and power decline during a fatigue run was less at 30°C (∼20–30%) than at 10°C (∼50%). The finding that the extent of fatigue is increased with shortening contractions and is lower at higher temperatures is consistent with the view that force depression by inorganic phosphate, which accumulates within fibers during activity, may be a primary cause of initial muscle fatigue.

A Mammalian Homolog of Drosophila Tumorous Imaginal Discs, Tid1, Mediates Agrin Signaling at the Neuromuscular Junction

Motoneuron-derived agrin clusters nicotinic acetylcholine receptors (AChRs) in mammalian muscle cells. We used two-hybrid screens to identify a protein, tumorous imaginal discs (Tid1), that binds to the cytoplasmic domain of muscle-specific kinase (MuSK), a major component of the agrin receptor. Like MuSK, Tid1 colocalizes with AChRs at developing, adult, and denervated motor endplates. Knockdown of Tid1 by short hairpin RNA (shRNA) in skeletal muscle fibers dispersed synaptic AChR clusters and impaired neuromuscular transmission. In cultured myotubes, Tid1 knockdown inhibited AChR clustering, as well as agrin-induced activation of the Rac and Rho small GTPases and tyrosine phosphorylation of the AChR, without affecting MuSK activation. Tid1 knockdown also decreased Dok-7-induced clustering of AChRs. Overexpression of the N-terminal half of Tid1 induced agrin- and MuSK-independent phosphorylation and clustering of AChRs. These results demonstrate that Tid1 is an essential component of the agrin signaling pathway, crucial for synaptic development.

Identification of a repeated domain within mammalian α-synemin that interacts directly with talin

The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, α-actinin, and talin. We report herein a direct interaction of human α-synemin with the cytoskeletal protein talin by protein–protein interaction assays. The 312 amino acid insert (SNTIII) present only within α-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human α- and β-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human α-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.

昆虫背脈管組織を用いた長期間室温で駆動するバイオアクチュエータの創製

Here we propose a bioactuator using an insect dorsal vessel (heart muscle) tissue which can drive autonomously at room temperature for a long term. Living muscle cells have been attracted much interest as an actuator because a muscle tissue is totally superior to artificial muscles. Previously reported bioactuators using mammalian heart muscle cells require precise environmental control to keep cells alive and contracting. On the other hand, an insect tissue is robust compared to mammalian cells; they can grow at room temperature for a long term without medium replacement. In this paper, we succeeded in driving micropillars by the dorsal vessel tissue for more than 90 days. The strongest displacement was 23 [μm] . Based on the results of displacement, the driving force was estimated 4.7 [μN] . The definite displacements more than 10 [μm] were observed for 58 days from the 15th day to the 72nd day. Therefore, the life cycle can be calculated at 7.5×105 times as the average frequency was about 0.15 [Hz] . These results suggest that the insect heart tissue is more promising material for a bioactuator at room temperature compared with the other biological cell based actuators.

細胞を用いたバイオアクチュエータによる生命機械システムの創製(基調講演2)

Living muscle cells have been attracted much interest as a novel actuator because they have high energy-conversion efficiency and can work only with chemical energy in glucose. Bioactuators using mammalian muscle tissues and cells were reported so far, however they require precise environmental control to keep them alive. We already proposed and demonstrated to utilize an insect dorsal vessel (heart muscle) tissue as an environmentally robust bioactuator. The actuator worked for more than 90 days at 25 ℃ without any maintenance.

2P2-J20 昆虫背脈管組織を用いたバイオアクチュエータの温度依存性の評価(ナノ・マイクロバイオシステム)

Living muscle cells have been attracted much interest as a novel actuator because they have high energy-conversion efficiency and can work only with chemical energy in glucose. Bioactuators using mammalian muscle tissues and cells were reported so far, however they require precise environmental control to keep them alive. We already proposed and demonstrated to utilize an insect dorsal vessel (heart muscle) tissue as an environmentally robust bioactuator. The actuator worked for more than 90 days at 25℃ without any maintenance. In this paper, the temperature dependence of the actuator was evaluated. The actuator was able to work at 5 to 40℃, but it was damaged irreversibly at 40℃. The Q10 (temperature coefficient from 20 to 30℃) of contraction frequency, force, and rate was 1.70, 0.77, and 1.28 respectively. These results suggest that the dorsal vessel is a less temperature-dependent bioactuator compared with mammalian cells.

Force generation examined by laser temperature‐jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

We examined the tension change induced by a rapid temperature jump (T-jump) in shortening and lengthening active muscle fibres. Experiments were done on segments of permeabilized single fibres (length (L0) ∼2 mm, sarcomere length 2.5 μm) from rabbit psoas muscle; [MgATP] was 4.6 mm, pH 7.1, ionic strength 200 mm and temperature ∼9°C. A fibre was maximally Ca2+-activated in the isometric state and a ∼3°C, rapid (< 0.2 ms), laser T-jump applied when the tension was approximately steady in the isometric state, or during ramp shortening or ramp lengthening at a limited range of velocities (0–0.2 L0 s−1). The tension increased to 2- to 3 × P0 (isometric force) during ramp lengthening at velocities > 0.05 L0 s−1, whereas the tension decreased to about < 0.5 × P0 during shortening at 0.1–0.2 L0 s−1; the unloaded shortening velocity was ∼1 L0 s−1 and the curvature of the force–shortening velocity relation was high (a/P0 ratio from Hill's equation of ∼0.05). In isometric state, a T-jump induced a tension rise of 15–20% to a new steady state; by curve fitting, the tension rise could be resolved into a fast (phase 2b, 40–50 s−1) and a slow (phase 3, 5–10 s−1) exponential component (as previously reported). During steady lengthening, a T-jump induced a small instantaneous drop in tension, followed by recovery, so that the final tension recorded with and without a T-jump was not significantly different; thus, a T-jump did not lead to a net increase of tension. During steady shortening, the T-jump induced a pronounced tension rise and both its amplitude and the rate (from a single exponential fit) increased with shortening velocity; at 0.1–0.2 L0 s−1, the extent of fibre shortening during the T-jump tension rise was estimated to be ∼1.2% L0 and it was shorter at lower velocities. At a given shortening velocity and over the temperature range of 8–30°C, the rate of T-jump tension rise increased with warming (Q10 ≈ 2.7), similar to phase 2b (endothermic force generation) in isometric muscle. Results are discussed in relation to the previous findings in isometric muscle fibres which showed that a T-jump promotes an early step in the crossbridge–ATPase cycle that generates force. In general, the finding that the T-jump effect on active muscle tension is pronounced during shortening, but is depressed/inhibited during lengthening, is consistent with the expectations from the Fenn effect that energy liberation (and acto-myosin ATPase rate) in muscle are increased during shortening and depressed/inhibited during lengthening.

Molecular basis of the phenotypic characteristics of mammalian muscle fibres.

Adult mammalian skeletal muscle fibres can be separated into two distinct groups, fast and slow. Within each group there is a continuum of metabolic enzyme activity levels. In addition there are fast and slow isoforms of various myofibrillar proteins such as myosin, tropomyosin and troponin. These proteins are multimeric and multiple isoforms of their subunits assemble to create a continuum of subtypes within each major group. Fibres which coexpress both fast and slow subunit isoforms have an increased number of possible isoform combinations such that an entire spectrum of fibre 'types' is found between the two extremes, fast and slow. Numerous myosin heavy chain and fast troponin T isoforms further increase the diversity of muscle fibres. Such cellular diversity helps to explain the dynamic nature of skeletal muscle. Each individual fibre is able to respond to various functional demands by appropriate changes in its phenotypic expression of specific proteins.

Movement of acid equivalents across the mammalian smooth muscle cell membrane.

Factors affecting intracellular pH (pHi) in the smooth muscle of guinea pig ureter have been investigated using pH-sensitive microelectrodes. Associated acids and bases appear to have free passage across the cell membrane but results suggest very low permeability to charged acid equivalents, thus implicating carrier-mediated movements in many of the observed pHi transients. Recovery from acidosis in the nominal absence of CO2 was inhibited by removal of Na+ and by the presence of amiloride, indicating that Na+/H+ exchange was responsible. The presence of CO2 resulted in a faster recovery from acidosis but, since intracellular buffering power was not increased, not a substantially faster effective extrusion of protons. Surprisingly, amiloride no longer caused discernable inhibition. Recovery from moderate acidosis remained Na+ dependent but was not inhibited by DIDS or acetazolamide or by the absence of Cl-, suggesting a dominant Na+-, and HCO3(-)-dependent mechanism unlike any hitherto described. Recovery from alkalosis was inhibited by DIDS and Cl(-)-free conditions, indicating that Cl-/HCO3- exchange was involved. Results suggest reversal of this mechanism on extreme acidosis. Experiments in vascular smooth muscle with fluorescent indicators confirm the presence of Na+/H+ exchange but provide conflicting evidence about the presence and properties of the HCO3(-)-dependent mechanism.

Comparison of the tension responses to ramp shortening and lengthening in intact mammalian muscle fibres: crossbridge and non-crossbridge contributions

We examined the tension responses to ramp shortening and lengthening over a range of velocities (0.1-5 L(0)/s) and at 20 degrees C and 30 degrees C in tetanized intact fibre bundles from a rat fast (flexor hallucis brevis) muscle; fibre length (L(0)) was 2.2 mm and sarcomere length approximately 2.5 microm. The tension change during ramp releases as well as ramp stretches showed an early transition (often appearing as an inflection) at 1-4 ms; the tension change at this transition and the length change at which it occurred increased with velocity. A second transition, indicated by a more gradual reduction in slope, occurred when the length had changed by 14-28 nm per half-sarcomere; the tension at this transition increased with lengthening velocity towards a plateau and it decreased with shortening velocity towards zero tension. The velocity dependence of the time to the transitions and the length change at the transitions showed some asymmetries between shortening and lengthening. Based on analyses of the velocity dependence of the tension and modelling, we propose that the first transition reflects the tension change associated with the crossbridge power stroke in shortening, or with the reversal of the power stroke in lengthening. Modelling shows that the reduction in slope at the second transition occurs when most of the crossbridges (myosin heads) that were attached at the start of the ramp become detached. After the second transition, the tension reaches a steady level in the model whereas the tension continues to increase during lengthening and continues to decrease during shortening in the experiments; this continuous tension change is seen at a wide range of initial sarcomere lengths and when active force is reduced by the myosin inhibitor, BTS. The continuous tension decline during shortening is not abolished by caffeine, but the rate of decline is reduced when the active force is depressed by BTS. We propose that stiffening of non-crossbridge visco-elastic elements upon activation contributes to the continuous tension rise during lengthening and the release of such tension and Ca-insensitive deactivation contribute to the tension decline during shortening in muscle fibres.

Embryonic motoneuron-skeletal muscle co-culture in a defined system

This paper describes a significant biotechnological advancement by creating a minimalist serum-free defined system to co-culture rat mammalian nerve and muscle cells in order to form functional neuromuscular junctions. To date, all the known in vitro nerve and muscle co-culture models use serum containing media; and while functional neuromuscular junctions (NMJ) are described, they failed to detail or quantify the minimum factors needed to recreate the NMJ in vitro. In this work, we demonstrate the development of a defined motoneuron and muscle co-culture system resulting in the formation of NMJs including: 1) a new culture technique, 2) a novel serum-free medium formulation and 3) a synthetic self-assembled monolayer (SAM) substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). We characterized the culture by morphology, immunocytochemistry, electrophysiology and videography. This model system provides a better understanding of the minimal growth factor and substrate interactions necessary for NMJ formation and provides a basic system that can be utilized for nerve–muscle tissue engineering, regenerative medicine and development of limb prosthetics.

Mitochondrial superoxide production in skeletal muscle fibers of the rat and decreased fiber excitability

Mammalian skeletal muscles generate marked amounts of superoxide (O(2)(.-)) at 37 degrees C, but it is not well understood which is the main source of O(2)(.-) production in the muscle fibers and how this interferes with muscle function. To answer these questions, O(2)(.-) production and twitch force responses were measured at 37 degrees C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O(2)(.-) production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O(2)(.-) production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O(2)(.-) accounts for most of the O(2)(.-) measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37 degrees C. Muscle fiber excitability at 37 degrees C was greatly improved in the presence of a membrane permeant O(2)(.-) dismutase mimetic (Tempol), demonstrating a direct link between O(2)(.-) production in the mitochondria and muscle fiber performance. This implicates mitochondrial O(2)(.-) production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell.

Reply from I. A. Telley, R. Stehle, K. W. Ranatunga, G. Pfitzer, E. Stüssi and J. Denoth

Reply from I. A. Telley, R. Stehle, K. W. Ranatunga, G. Pfitzer, E. Stüssi and J. Denoth

Calcium Release Domains in Mammalian Skeletal Muscle Studied with Two-photon Imaging and Spot Detection Techniques

The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks (∼23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca2+ transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest ΔF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca2+ release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in ∼1.4 ms and dissipate within ∼4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca2+ transients peaked at T-tubule locations, was 0.62 μm, similar to the 0.61 μm measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca2+ domains in adult mammalian muscle fibers are confined to Ca2+ release sites located at the junctional sarcoplasmic reticulum (SR).

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Prolonged High-Pressure Treatments in Mammalian Skeletal Muscle Result in Loss of Functional Sodium Channels and Altered Calcium Channel Kinetics

Activation and inactivation of ion channels involve volume changes from conformational rearrangements of channel proteins. These volume changes are highly susceptible to changes in ambient pressure. Depending on the pressure level, channel function may be irreversibly altered by pressure. The corresponding structural changes persist through the post-decompression phase. High-pressure applications are a useful tool to evaluate the pressure dependence as well as pressure limits for reversibility of such alterations. Mammalian cells are only able to tolerate much lower pressures than microorganisms. Although some limits for pressure tolerance in mammalian cells have been evaluated, the mechanisms of pressure-induced alteration of membrane physiology, in particular of channel function, are unknown. To address this question, we recorded fast inward sodium (I(Na)) and slowly activating L-type calcium (I(Ca)) currents in single mammalian muscle fibers in the post-decompression phase after a prolonged 3-h, high-pressure treatment of up to 20 MPa. I(Na) and I(Ca) peak amplitudes were markedly reduced after pressure treatment at 20 MPa. This was not from a general breakdown of membrane integrity as judged from in situ high-pressure fluorescence microscopy. Membrane integrity was preserved even for pressures as high as 35 MPa at least for pressure applications of shorter durations. Therefore, the underlying mechanisms for the observed amplitude reductions have to be determined from the activation (time-to-peak [TTP]) and inactivation (tau(dec)) kinetics of I(Na) and I(Ca). No major changes in I(Na) kinetics, but marked increases, both in TTP and tau(dec) for I(Ca), were detected after 20 MPa. The apparent molecular volume changes (activation volumes) deltaV(double dagger) for the pressure-dependent irreversible alteration of channel gating approached zero for Na+ channels. For Ca2+ channels, deltaV(double dagger) was very large, with approx 2.5-fold greater values for channel activation than inactivation (approx 210 A3). We conclude, that in skeletal muscle, high pressure differentially and irreversibly affects the gating properties and the density of functional Na+ and Ca2+ channels. Based on these results, a model of high pressure-induced alterations to the channel conformation is proposed.

Parvalbumin deficiency in fast‐twitch muscles leads to increased ‘slow‐twitch type’ mitochondria, but does not affect the expression of fiber specific proteins

Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a 'slow-onset' Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV-/- mice. Unexpectedly, PV-/- fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV-/- fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slow-twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit beta revealed the PV-/- tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fast-twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast- and slow-twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca2+ transients in PV-/- muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin- and Ca2+/calmodulin-dependent kinase II-dependent pathways. We propose that 'slow-twitch type' mitochondria in PV-/- fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca2+ removal.

Optical Imaging and Functional Characterization of the Transverse Tubular System of Mammalian Muscle Fibers using the Potentiometric Indicator di-8-ANEPPS

Potentiometric dyes are useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes, the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in muscle fibers stretched to attain sarcomere lengths of up to 3.5 microm. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530-550 nm band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.